U.S. patent application number 16/046118 was filed with the patent office on 2019-01-17 for enhanced expression of human or humanized immunoglobulin in non-human transgenic animals.
The applicant listed for this patent is THERAPEUTIC HUMAN POLYCLONALS INC.. Invention is credited to Roland Buelow.
Application Number | 20190017021 16/046118 |
Document ID | / |
Family ID | 40382388 |
Filed Date | 2019-01-17 |
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United States Patent
Application |
20190017021 |
Kind Code |
A1 |
Buelow; Roland |
January 17, 2019 |
ENHANCED EXPRESSION OF HUMAN OR HUMANIZED IMMUNOGLOBULIN IN
NON-HUMAN TRANSGENIC ANIMALS
Abstract
The present invention describes transgenic animals with
human(ized) immunoglobulin loci and transgenes encoding human(ized)
Ig.alpha. and/or Ig.beta. sequences. Of particular interest are
animals with transgenic heavy and light chain immunoglobulin loci
capable of producing a diversified human(ized) antibody repertoire
that have their endogenous production of Ig and/or endogenous
Ig.alpha. and/or Ig.beta. sequences suppressed. Simultaneous
expression of human(ized) immunoglobulin and human(ized) Ig.alpha.
and/or Ig.beta. results in normal B-cell development, affinity
maturation and efficient expression of human(ized) antibodies.
Inventors: |
Buelow; Roland; (Palo Alto,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THERAPEUTIC HUMAN POLYCLONALS INC. |
MOUNTAIN VIEW |
CA |
US |
|
|
Family ID: |
40382388 |
Appl. No.: |
16/046118 |
Filed: |
July 26, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15272051 |
Sep 21, 2016 |
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16046118 |
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12972290 |
Dec 17, 2010 |
9476027 |
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15272051 |
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11895910 |
Aug 27, 2007 |
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12972290 |
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60841890 |
Sep 1, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/70503 20130101;
A01K 2267/01 20130101; A61P 29/00 20180101; A01K 67/0275 20130101;
C07H 21/04 20130101; A01K 2217/072 20130101; C12N 2510/02 20130101;
C12N 5/0635 20130101; C12N 2510/04 20130101; A61K 39/00 20130101;
C09K 5/045 20130101; C09K 2205/126 20130101; A01K 67/0278 20130101;
A01K 67/0276 20130101; A01K 2227/107 20130101; C12N 2517/02
20130101; A61P 35/00 20180101; A01K 2207/15 20130101; C12N 15/85
20130101; A01K 2217/052 20130101; A01K 2217/054 20130101; C12N
15/8509 20130101; C12N 15/79 20130101 |
International
Class: |
C12N 5/0781 20100101
C12N005/0781; C12N 15/85 20060101 C12N015/85; A01K 67/027 20060101
A01K067/027; C07K 14/705 20060101 C07K014/705; C09K 5/04 20060101
C09K005/04; A61K 39/00 20060101 A61K039/00 |
Claims
1-6. (canceled)
7. A method for producing human or humanized antibodies in a
non-human animal, comprising the steps of: (a) introducing and
expressing a transgene construct encoding either a native human
Ig.alpha. subunit or a chimeric Ig.alpha. subunit, and/or a
transgene construct encoding either a native human Ig.beta. subunit
or a chimeric Ig.beta. subunit into the non-human animal; (b)
introducing and expressing a transgene construct encoding a human
or humanized immunoglobulin locus into the non-human animal; (c)
subjecting the animal to an antigenic stimulus; and (d) isolating
human or humanized antibodies from the animal.
8. The method according to claim 7, wherein the antibody is a
monoclonal antibody.
9. The method according to claim 7, wherein the antibody is a
fragment of a monoclonal antibody.
10. The method according to claim 9, wherein the antibody fragment
is fused to a heterologous amino acid sequence.
11. An isolated human or humanized antibody produced with the
method according to claim 1.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a non-provisional application filed under 37 CFR
1.53(b), claiming priority under U.S.C. Section 119(e) to U.S.
Provisional Patent Application Ser. No. 60/841,980 filed Sep. 1,
2006.
FIELD OF THE INVENTION
[0002] This invention relates to a method to improve the expression
of human(ized) immunoglobulin in non-human transgenic animals by
promoting normal B-cell development and by sustaining the
expression of human(ized) antibodies in non-human animals harboring
human(ized) immunoglobulin loci. In particular, this invention
relates to the simultaneous expression of transgenes encoding
human(ized) Ig.alpha. and/or I.beta., components of the B-cell
receptor, and transgenes encoding a human(ized) immunoglobulin
locus or loci. This method allows for the dominant expression of
human(ized) antibodies, for example in the blood, milk or eggs of
the transgenic non-human animals.
DESCRIPTION OF THE RELATED ART
[0003] Antibodies are an important class of pharmaceutical products
that have been successfully used in the treatment of various human
diseases and conditions, such as cancer, allergic diseases,
prevention of transplant rejection and host-versus-graft
disease.
[0004] A major problem of the antibody preparations obtained from
non-human animals is the intrinsic immunogenicity of non-human
immunoglobulins in human patients. In order to reduce the
immunogenicity of non-human antibodies, it has been shown that by
fusing animal variable (V) region exons with human constant (C)
region exons, a chimeric antibody gene can be obtained. Such
chimeric or humanized antibodies have minimal immunogenicity to
humans and are appropriate for use in the therapeutic treatment of
human subjects.
[0005] Humanized monoclonal antibodies have been developed and are
in clinical use. However, the use of monoclonal antibodies in
general, whether chimeric, humanized or human, for the treatment of
devastating diseases such as cancer or infections with virulent
pathogens, is limited due to the complexity, multifactorial
etiology and adaptivity of these diseases. Monoclonal antibodies
directed against singularly defined targets usually fail when those
targets change, evolve and mutate. For instance, malignancies may
gain resistance to standard monoclonal antibody therapies. A
solution to this problem is to use polyclonal antibodies which have
the ability to target a plurality of evolving targets. Polyclonal
antibodies can neutralize bacterial or viral toxins, and direct
immune responses to kill and eliminate pathogens.
[0006] Accordingly, there is a great clinical need for suitable
methods for the large-scale production of high-titer,
high-affinity, humanized polyclonal and monoclonal antibodies.
Further, since production of antibodies in larger transgenic
animals like rabbits, chickens, sheep and cows is favored from the
standpoint of antibody yield, creation of larger founder animals
expressing higher amounts of transgene-encoded products is also
highly desirable.
[0007] Humanized monoclonal antibodies are typically human
antibodies in which some CDR residues, and possibly some FR
residues, are substituted by residues from analogous sites in
non-human, animal, e.g. rodent, antibodies. Humanization can be
essentially performed following the method of Winter and co-workers
(Jones et al., Nature, 321: 522 (1986); Riechmann et al., Nature,
332: 323 (1988); Verhoeyen et al., Science, 239: 1534 (1988)), by
substituting non-human, animal CDRs or CDR sequences (e.g. rodent),
for the corresponding sequences of a human monoclonal antibody.
[0008] While making humanized antibodies in animals, one problem
encountered is the endogenous production of host antibody over
transgenic antibody, which needs to be suppressed. It has been
described that the homozygous deletion of the antibody, heavy-chain
joining region (JH) gene in chimeric and germ-line mutant mice,
results in the complete inhibition of endogenous antibody
production. Transfer of a human germ-line immunoglobulin gene array
into such germ-line mutant mice will result in the production of
human antibodies upon antigen challenge. See, e.g., Jakobovits et
al., Proc. Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al.,
Nature, 362: 255 (1993); Bruggemann et al., Year in Immunol., 7: 33
(1993); U.S. Pat. No. 7,064,244 issued Jun. 20, 2006; the
disclosures of which are incorporated herein by reference in their
entirety.
[0009] The introduction of human immunoglobulin genes into the
genome of mice results in the expression of a diversified human
antibody repertoire in these genetically engineered mice. The
generation of mice expressing human-mouse chimeric antibodies has
been described by Pluschke et al., Journal of Immunological Methods
215: 27-37 (1998). The generation of mice expressing human
immunoglobulin polypeptides has been described by Neuberger et al.,
Nature 338: 350-2 (1989); Lonberg et al., Int. Rev. Immunol.
13(1):65-93 (1995); and Bruggemann et al., Curr. Opin. Biotechnol.,
8(4): 455-8 (1997); U.S. Pat. No. 5,545,806, issued August 1996;
U.S. Pat. No. 5,545,807, issued August 1996 and U.S. Pat. No.
5,569,825, issued October 1996; the disclosures of which are
incorporated herein by reference in their entirety. The generation
of cows expressing human antibodies has been described by Kuroiwa
et al., Nature Biotech 20(9): 889-894 (2002). The production of
non-human transgenic animals expressing human(ized) immunoglobulin
transloci and the production of antibodies from such transgenic
animals have also been described in detail in PCT Publication Nos.
WO 92/03918, WO 02/12437, and in U.S. Pat. Nos. 5,814,318, and
5,570,429, the disclosures of which are hereby expressly
incorporated by reference in their entirety. The humanized
antibodies obtained have minimal immunogenicity to humans and are
appropriate for use in the therapeutic treatment of human
subjects.
[0010] While the genetic engineering approaches cited above result
in the expression of human immunoglobulin polypeptides in
genetically engineered mice, the level of human immunoglobulin
expression is lower than normal. This may be due to
species-specific regulatory elements in the immunoglobulin loci
that are necessary for efficient expression of immunoglobulins. As
demonstrated in transfected cell lines, regulatory elements present
in human immunoglobulin genes may not function properly in
non-human animals. Several regulatory elements in immunoglobulin
genes have been described. Of particular importance are enhancers
downstream (3') of heavy chain constant regions and intronic
enhancers in light chain genes. In addition, other, yet to be
identified, control elements may be present in immunoglobulin
genes. Studies in mice have shown that the membrane and cytoplasmic
tail of the membrane form of immunoglobulin molecules play an
important role in expression levels of human-mouse chimeric
antibodies in the serum of mice homozygous for the human C.gamma.1
gene. Therefore, for the expression of heterologous immunoglobulin
genes in animals, it is desirable to replace sequences that contain
enhancer elements and exons encoding transmembrane (M1 exon) and
cytoplasmic tail (M2 exon) with sequences that are normally found
in the animal in similar positions.
[0011] Human immunoglobulin expression in these genetically
engineered animals may also be affected by B-cell development of
the non-human B-cells carrying the human or humanized
immunoglobulin loci. The influence of the B-cell receptor (BCR) on
B-cell development has been studied extensively in mice. However,
it has been unclear how a human or partially human antibody
combines to form a functional BCR, and whether such a BCR would
efficiently influence the development and survival of non-human
B-cells expressing human(ized) Ig, in transgenic animals, which, in
turn, would affect antibody yields.
BRIEF DESCRIPTION OF DRAWINGS
[0012] FIG. 1 shows an amino acid alignment of the human Ig.alpha.
polypeptide sequence (SEQ ID NO: 1) with other non-human Ig.alpha.
sequences (SEQ ID NOs: 2-6).
[0013] FIG. 2 shows an amino acid alignment of the human Ig.beta.
polypeptide sequence (SEQ ID NO: 7) with other non-human IG.beta.
sequences (SEQ ID NOs: 8-12).
SUMMARY OF THE INVENTION
[0014] In one aspect, the invention provides a transgene construct
encoding a chimeric Ig.alpha. subunit of the BCR, wherein the
chimeric Ig.alpha. subunit comprises an intracellular domain
sequence and a transmembrane domain sequence of a non-human,
Ig.alpha. polypeptide sequence; and further, a polypeptide having
at least 85% sequence identity to the extracellular domain of human
Ig.alpha. of SEQ ID NO.: 1.
[0015] In a second aspect, the invention provides a transgene
construct encoding a chimeric Ig.beta. subunit of the BCR, wherein
the chimeric I.beta. subunit comprises an intracellular domain
sequence and a transmembrane domain sequence of a non-human,
Ig.beta. polypeptide sequence, and further, a polypeptide having at
least 85% sequence identity to the extracellular domain of the
human Ig.beta. of SEQ ID NO.:7.
[0016] In a certain embodiment of the invention, the type of
non-human Ig.alpha. polypeptide sequence includes, but is not
limited to, the bovine (SEQ ID NO: 2); murine (SEQ ID NO: 3);
canine (SEQ ID NO: 4); primate ((SEQ ID NO: 5); rabbit (SEQ ID NO:
6) or other non-human sequences. In another embodiment of the
invention, the type of non-human Ig.beta. polypeptide sequence
includes, but is not limited to, the canine (SEQ ID NO: 8); rat
(SEQ ID NO: 9); bovine (SEQ ID NO: 10); murine (SEQ ID NO: 11);
chicken (SEQ ID NO: 12) or other non-human sequences.
[0017] In a third aspect, the non-human transgenic animal comprises
(a) a transgene construct encoding either a full-length, human
Ig.alpha. subunit of SEQ ID NO.: 1, or the chimeric Ig.alpha.
subunit as defined above, and/or, (b) a transgene construct
encoding either a full-length, human Ig.beta. subunit of SEQ ID
NO.: 7, or the chimeric Ig.beta. subunit, as defined above, and,
(c) a transgene construct encoding a human(ized) immunoglobulin
locus, wherein the resultant transgene products combine to form a
human(ized) B-cell receptor complex.
[0018] In one embodiment of this aspect, the expression of any
endogenous Ig production, and/or, endogenous Ig.alpha. and/or
endogenous Ig.beta. subunit expression, of the non-human transgenic
animal is substantially reduced.
[0019] In another embodiment, the non-human transgenic animal is
selected from a group consisting of rabbit, mouse, rat, pig, sheep,
goat, bird, horse, donkey and cow. In a preferred embodiment, the
non-human transgenic animal is a rabbit.
[0020] In a fourth aspect, the invention also provides an isolated
human(ized) immunoglobulin from the non-human transgenic animal
defined above, which is either an antibody or an antibody fragment.
In a certain embodiment of this aspect, the isolated human(ized)
immunoglobulin is either a polyclonal or a monoclonal antibody, or
alternately, is an antibody fragment. The antibody fragment can be
either from a polyclonal or a monoclonal antibody. Further, the
antibody or the antibody fragment can be labeled, or fused to a
toxin to form an immunotoxin, or coupled to a therapeutic agent, or
fused to any heterologous amino acid sequence well-defined and used
in the art. In some embodiments, the antibody fragment is a Fc, Fv,
Fab, Fab' or F(ab').sub.2 fragment.
[0021] In a fifth aspect, the invention provides an isolated B-cell
from the non-human transgenic animal defined above, where the
B-cell expresses either the native human Ig.alpha. subunit or a
chimeric Ig.alpha. subunit and/or either the native human I.beta.
subunit or the chimeric Ig.beta. subunit, and further, also
expresses the human(ized) immunoglobulin locus. In certain
embodiments, this B-cell is immortalized and in a preferred
embodiment, is derived from a rabbit.
[0022] In a sixth aspect, the invention provides an antibody
preparation comprising an antibody or an antibody fragment, as
described above.
[0023] In a seventh aspect, the invention provides a pharmaceutical
composition comprising an antibody or antibody fragment, as
described above, in a mixture with a pharmaceutically acceptable
ingredient. The pharmaceutical composition can comprise either a
monoclonal antibody or a fragment thereof, or, one or a plurality
of polyclonal antibodies or fragments thereof.
[0024] In an eighth aspect, the invention provides a method for
producing human(ized) antibodies in a non-human animal comprising:
(a) introducing and expressing a transgene construct encoding
either a native human Ig.alpha. subunit or a chimeric Ig.alpha.
subunit, and/or a transgene construct encoding either a native
human Ig.beta. subunit or a chimeric Ig.beta. subunit into the
non-human animal; and, (b) introducing and expressing a transgene
construct encoding a human(ized) immunoglobulin locus into the
non-human animal; (c) subjecting the animal to an antigenic
stimulus; and (d) isolating human(ized) antibodies from the animal.
In a certain embodiment of this aspect, the antibody is either a
polyclonal or a monoclonal antibody, or is a fragment of a
polyclonal or a monoclonal antibody. Further, the antibody or
antibody fragment can either be labeled, or can be fused to a toxin
to form an immunotoxin, or coupled to a therapeutic agent, or can
be fused to any heterologous amino acid sequence.
[0025] In a ninth aspect, the invention provides a method for
producing a non-human animal expressing human(ized) antibodies
comprising: (a) introducing and expressing a transgene construct
encoding either a native human Ig.alpha. subunit or a chimeric
Ig.alpha. subunit and/or a transgene construct encoding either a
native human Ig.beta. subunit or a chimeric Ig.beta. subunit into
the B-cell of the non-human animal; and, (b) introducing and
expressing a transgene construct encoding a human(ized)
immunoglobulin locus into the non-human animal; wherein the
resultant transgene products combine to form a human(ized) B-cell
receptor complex. In one embodiment, the non-human animal
expressing human(ized) antibodies is an animal that creates
antibody diversity by gene conversion and/or somatic hypermutation.
In a preferred embodiment, the animal is a rabbit.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0026] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
Singleton et al., Dictionary of Microbiology and Molecular Biology
2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March,
Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th
ed., John Wiley & Sons (New York, N.Y. 1992), provide one
skilled in the art with a general guide to many of the terms used
in the present application.
[0027] One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could be used in the practice of the present invention. Indeed, the
present invention is in no way limited to the methods and materials
described. For purposes of the present invention, the following
terms are defined below.
[0028] "A transgene construct or expression construct" as defined
herein, refers to a DNA molecule which contains the coding sequence
for at least one transgene of interest along with appropriate
regulatory sequences required for temporal, cell specific and/or
enhanced expression of the transgene(s) of interest within target
cells of a non-human transgenic animal.
[0029] "B-cells" are defined as B-lineage cells that are capable of
undergoing rearrangement of immunoglobulin gene segments and
expressing immunoglobulin genes at some stage in their life cycle.
These cells include, but are not limited to, early pro-B-cells,
late pro-B-cells, large pre-B-cells, small pre-B-cells, immature
B-cells, mature B-cells, memory B-cells, plasma cells, etc.
[0030] "B-cell receptor (BCR) complex" as defined herein, refers to
the multisubunit immune recognition receptor expressed on B-cells,
which includes the following subunits: the antigen (Ag) receptor,
the membrane-bound immunoglobulin (mIg), the Ig.alpha. subunit and
the Ig.beta. subunit. The B-cell receptor, its components, and its
association with the five immunoglobulin classes have been
described by Wienands et al., EMBO J. 9(2): 449-455 (1990),
Venkitaraman et al., Nature 352: 777-781 (1991), Herren et al.,
Immunologic Res. 26(1-3): 35-43 (2002). In addition, there are
several BCR-associated proteins (BAPs) that have been cloned and
sequenced, but their function(s) remain unknown, and their role, as
components of the BCR, has been questioned. BCR-associated proteins
have been described by Adachi et al., EMBO J 15(7): 1534-1541
(1996) and Schamel et al., PNAS 100(17): 9861-9866 (2003).
[0031] "Native Ig.alpha. or Ig.beta. subunits" refer to naturally
occurring Ig.alpha. or .beta. polypeptide sequences, which include
naturally occurring alleles of Ig.alpha. or Ig.beta. subunits found
in a given type of animal, or in a related species. These are also
referred to as "full-length Ig.alpha. or Ig.beta. sequences". The
human Ig.alpha. polypeptide sequence was cloned by Flaswinkel et
al., Immunogenetics 36 (4): 266-69 (1992); Accession number M74721
(FIG. 1, SEQ ID NO: 1). The human Ig.beta. polypeptide sequence was
cloned by Mueller et al., Eur. J. Biochem. 22, 1621-25 (1992);
Accession number M80461 (FIG. 2, SEQ ID NO: 7).
[0032] The term "human(ized)" refers to an entirely human sequence
or a sequence containing one or more human sequences. Thus, the
term, as used herein, includes human and humanized sequences.
[0033] A "chimeric Ig.alpha." subunit or protein or polypeptide
refers to an Ig.alpha. polypeptide sequence from an animal (e.g.;
rat, mouse, human, rabbit, chicken, etc.), in which one or more
domains of the Ig.alpha. polypeptide are replaced with a
corresponding domain or domains from a different Ig.alpha.
polypeptide of another animal or species, or with a corresponding
domain or domains from a different allelic Ig.alpha. version, or
from a variant Ig.alpha. sequence with one or more amino acid
substitutions, or from a variant Ig.alpha. sequence having at least
85% sequence identity to the corresponding domain of a given
Ig.alpha. sequence. The terms "chimeric Ig.alpha." and "human(ized)
Ig.alpha." are used interchangeably throughout the specification.
Ig.alpha. polypeptide sequences (SEQ ID NOs: 2-6) from some
non-human animals are also defined in FIG. 1.
[0034] A "chimeric Ig.beta." subunit or protein or polypeptide
refers to an Ig.beta. polypeptide sequence from an animal (e.g.;
rat, mouse, human, rabbit, chicken, etc.), in which one or more
domains of the I.beta. polypeptide are replaced with a
corresponding domain or domains from a different Ig.beta.
polypeptide of another animal or species, or with a corresponding
domain or domains from a different allelic Ig.beta. version, or
from a variant Ig.beta. sequence with one or more amino acid
substitutions, or from a variant Ig.beta. sequence having at least
85% sequence identity to the corresponding domain of a given
Ig.beta. sequence. The terms "chimeric Ig.beta." and "human(ized)
Ig.beta." are used interchangeably throughout the specification.
Ig.beta. polypeptide sequences (SEQ ID NOs: 8-12) from some
non-human animals are defined in FIG. 2.
[0035] "Intracellular polypeptide or domain" or "cytoplasmic tail"
refers to that part of the polypeptide sequence of a given
membrane-bound protein or subunit that exists within the bounds of
the cell. Usually, the intracellular domain of the protein is
responsible for signal transduction.
[0036] By "intracellular domain sequence" of an Ig.alpha. or
Ig.beta. subunit is meant the polypeptide sequence of the Ig.alpha.
or Ig.beta. polypeptide, or fragments thereof, that usually exist
within the bounds of the cell.
[0037] "Transmembrane domain sequence" of an Ig.alpha. or Ig.beta.
subunit is meant the polypeptide sequence of the Ig.alpha. or
I.beta. polypeptide, or fragments thereof that spans a biological
membrane such as a plasma membrane, organelle membrane, or lipid
bilayer. The "transmembrane domain sequence" as defined herein
includes naturally occurring membrane-spanning polypeptides, or can
be non-naturally occurring consensus sequences, or fragments
thereof.
[0038] "Extracellular polypeptide or domain" refers to that part of
the polypeptide sequence of a given membrane-bound protein or
subunit that usually exists outside the bounds of the cell. By
"extracellular domain of Ig.alpha. or Ig.beta." is meant the
polypeptide sequence of the Ig.alpha. or Ig.beta. polypeptide, or
fragments thereof, that exist outside the bounds of the cell.
[0039] The term "human(ized) immunoglobulin locus" as used herein
includes both naturally occurring sequences of a human
immunoglobulin or Ig gene locus or a segment thereof, degenerate
forms of naturally occurring sequences of a human Ig gene locus or
segments thereof, as well as synthetic sequences that encode a
polypeptide sequence substantially identical to a polypeptide
encoded by a naturally occurring sequence of a human Ig gene locus
or a segment thereof. In a particular embodiment, the human Ig gene
segment renders the immunoglobulin molecule non-immunogenic in
humans. Here, the terms "human(ized) or humanized immunoglobulin
(Ig) heavy and/or light chain locus" or "human or human(ized)
immunoglobulin or Ig locus" are used interchangeably.
[0040] The term "human(ized) B-cell receptor (BCR) complex" as used
herein refers to those multisubunit BCR complexes in which the
Ig.alpha. subunit is either a native, human Ig.alpha. subunit or a
chimeric Ig.alpha. subunit having human or humanized Ig.alpha.
sequences as described above; and/or further, in which the Ig.beta.
subunit is either a native, human Ig.beta. subunit or a chimeric
Ig.beta. subunit having human or humanized Ig.beta. sequences as
described above; and further, where the membrane-bound
immunoglobulin (mIg) is that of a human(ized) immunoglobulin, as
described above.
[0041] The terms "human antibody" and "human immunoglobulin" are
used herein to refer to antibodies and immunoglobulin molecules
comprising fully human sequences.
[0042] The terms "humanized antibody" and "humanized
immunoglobulin," as used herein, mean an immunoglobulin molecule
comprising at least a portion of a human immunoglobulin polypeptide
sequence (or a polypeptide sequence encoded by a human
immunoglobulin gene segment). The humanized immunoglobulin
molecules of the present invention can be isolated from a
transgenic non-human animal engineered to produce humanized
immunoglobulin molecules. Such humanized immunoglobulin molecules
are less immunogenic to primates, especially humans, relative to
non-humanized immunoglobulin molecules prepared from the animal or
prepared from cells derived from the animal. Humanized
immunoglobulins or antibodies include immunoglobulins (Igs) and
antibodies that are further diversified through gene conversion and
somatic hypermutations in gene converting animals. Such humanized
Ig or antibodies are not "human" since they are not naturally made
by humans (since humans do not diversify their antibody repertoire
through gene conversion) and yet, the humanized Ig or antibodies
are not immunogenic to humans since they have human Ig sequences in
their structure.
[0043] By the term " substantially reduced" endogenous Ig
production, and/or Ig.alpha. and/or Ig.beta. subunits expression is
meant that the degree of production of either the endogenous Ig
alone or additionally, endogeous Ig.alpha. and/or Ig.beta.
expression is reduced preferably at least about 30%-49%, or more
preferably at least about 50%-79%, or even more preferably at least
about 80-89%, or most preferably by about 90-100% in the transgenic
animal.
[0044] The term "monoclonal antibody" is used to refer to an
antibody molecule synthesized by a single clone of B-cells.
[0045] The term "polyclonal antibody" is used to refer to a
population of antibody molecules synthesized by a population of
B-cells.
[0046] An "immunoglobulin (Ig) locus" having the capacity to
undergo gene rearrangement and gene conversion is also referred to
herein as a "functional" Ig locus, and the antibodies with a
diversity generated by a functional Ig locus are also referred to
herein as "functional" antibodies or a "functional" repertoire of
antibodies.
[0047] The term "non-human (transgenic) animal" as used herein
includes, but is not limited to, mammals such as, for example,
non-human primates, rodents (e.g. mice and rats), non-rodent
mammals, such as, for example, rabbits, pigs, sheep, goats, cows,
pigs, horses and donkeys, and birds (e.g., chickens, turkeys,
ducks, geese and the like). The term "non-primate animal" as used
herein includes, but is not limited to, mammals other than
primates, including but not limited to the mammals specifically
listed above.
DETAILED DESCRIPTION
[0048] This invention is based, at least in part, on the
recognition that the production of human or humanized
immunoglobulin (including immunoglobulin chains) in a non-human
transgenic animal can be significantly increased by co-expressing
human or humanized Ig.alpha. and/or Ig.beta. in the B cells of the
animal. The inclusion of human or humanized Ig.alpha. and/or
Ig.beta. in the B cells in transgenic animals is believed to
reconstitute and improve interactions between the B-cell receptor
proteins, thereby enhancing antigen recognition, B-cell development
and survival of the B cells carrying such transgenes. The
co-expression of humanized immunoglobulin in transgenic animals
already carrying the human or humanized Ig.alpha. and/or Ig.beta.
transgenes would vastly improve humanized immunoglobulin
production. It would be additionally desirable to express both, the
human or humanized Ig.alpha. and/or Ig.beta. transgene and the
humanized immunoglobulin transgene against a knockout background
of, preferably, both endogenous Ig, as well as endogenous Ig.alpha.
and/or Ig.beta..
The B-Cell Receptor and Its Associated Proteins
[0049] The B-cell receptor consists of membrane bound
immunoglobulin and a signal-transducing heterodimer, consisting of
two disulfide-linked glyoproteins called Ig.alpha. and Ig.beta.. In
addition, BCR associated proteins (BAPs) have been described.
[0050] Expression of the BCR is important for B-cell development,
selection and survival. These processes depend on BCR signaling
through the Ig.alpha./Ig.beta. heterodimer. The cytoplasmic domains
of these molecules carry a sequence motif that contains several
tyrosine residues assembled into the so-called immunoreceptor
tyrosine-based activation motif (ITAM), which are phosphorylated
upon BCR triggering.
[0051] Gene targeting experiments have shown that the cytoplasmic
domains of the Ig.alpha./Ig.beta. heterodimer are crucial for
B-cell development. Signals transduced by the Ig.alpha./Ig.beta.
heterodimer are involved in both positive and negative selection of
developing B-cells.
[0052] A membrane bound immunoglobulin (mIg) molecule consists of
two heavy chains, forming a homodimer, and two light chains, each
of which is covalently bound to one of the heavy chains. At the
N-terminus the heavy chain carries a VH domain, which, depending on
the isotype, is followed by either 4 (IgM, IgE), 3 (IgG, IgA) or 2
(IgD) C-domains. The antigen-binding site is formed by the
hypervariable regions of a VH:VL pair. Thus, each mIg molecule has
two antigen binding sites.
[0053] The mIgM molecule differs from the secreted form of IgM in
that the secreted IgM forms a pentamer with 10 potential antigen
binding sites. The pentamerization is controlled by sequences in
the C-terminal part of the secreted .mu.s chain. This part,
consisting of 22 amino acids, is absent in the membrane-bound .mu.m
chain, which instead carries 48 C-terminal amino acids encoded by
the M1 and M2 exons.
[0054] The gm-specific part of the sequence is the most
evolutionarily conserved part of the whole IgM molecule. It is
nearly identical between mouse, rabbit and human mIgM, and the
conservation is still obvious if one compares mouse with shark
mIgM. Conservation of amino acids is also apparent when one
compares the C-terminal sequence of mIgM to that of other mIg
isotypes of the mouse. This finding provides evidence that the
conserved transmembrane amino acids are interacting either with
each other in the H chain homodimer or with the Ig.alpha. and
Ig.beta. subunits.
[0055] Both, Ig.alpha. and Ig.beta. have a 22 amino acid
transmembrane segment, followed by a C-terminal cytoplasmic tail of
about 40-70 amino acids, which contain several tyrosine residues.
At the N-terminus, both proteins carry a leader peptide, followed
by a extracellular domain containing cysteine residues, a
tryptophan, as well as several other conserved amino acids found in
proteins of the Ig superfamily. This suggests that the
extracellular parts of both Ig.alpha. and Ig.beta. subunits form an
Ig-like domain. Besides cysteines that form intra-domain disulfide
bonds, the Ig.alpha. and Ig.beta. sequences contain additional
cysteines that presumably form inter-chain disulfide bonds between
the Ig.alpha. and Ig.beta. subunits.
[0056] A comparison between the mouse and the human Ig.alpha.
sequence shows that, all residues important for the information of
the Ig domain and inter-chain bonds are conserved between the
Ig.alpha. of the two species. The comparison, however, also shows
that sequence conservation in the extracellular part only amounts
to about 56%, while the transmembrane and cytoplasmic tail show
conservation of 100% and 87%, respectively. The latter reflects the
importance of the residues within the C-terminal part of the
molecule.
[0057] The assembly of the mIgM molecule with the
Ig.alpha./Ig.beta. heterodimer is necessary for surface expression
of mIgM. This requirement can be abolished by mutations of the
transmembrane part of the pm chain. For example, replacement of the
transmembrane region of the .mu.m chain with the transmembrane part
of the H-2K.sup..kappa. molecule results in the surface expression
of mIgM independent of Ig.alpha./Ig.beta.. These data demonstrate
that the .mu.m transmembrane region is required for specific
interactions between the .mu.m chain and the Ig.alpha./Ig.beta.
heterodimer. In addition, B-cells have a control mechanism that
prevents transport of single or incompletely assembled components
of transmembrane protein complex out of the ER.
[0058] Although the transmembrane portions of the BCR are probably
the most important structures that are required for the formation
of the BCR complex, the extracellular Ig-domain of Ig.alpha. and
Ig.beta. has also been suggested to play a role in the binding of
the mIgM molecule. For instance, in the mouse cell line J558L
.mu.m, which does not express mouse Ig.alpha., transfection with an
Ig.alpha. transgene restored the surface expression of mIgM.
Interestingly, transfection with a mouse Ig.alpha. gene resulted in
10-times higher expression than transfection with a human Ig.alpha.
gene. This data suggests that the extracellular domain of
Ig.alpha., additionally, may interact with the extracellular parts
of mIgM. On the other hand, transgenic mice with human
immunoglobulin loci do express human immunoglobulins. It remains
unclear whether B-cell development, B-cell survival or expression
of human(ized) mIgM in transgenic non-human animals would be
influenced by the co-expression of human Ig.alpha. and/or human
Ig.beta. in B-cells carrying human(ized) mIgM genes.
[0059] In addition, there are several BCR-associated proteins
(BAPs) that have been cloned and sequenced, but their function(s)
remain unknown. Even though these proteins are associated with the
BCR, their role, as components of the BCR, has been questioned.
Yet, the ubiquitous expression and strong evolutionary conservation
of BAPs suggest that they must play an important role, possibly in
general cellular processes and several putative functions have been
proposed. For example, these proteins may be involved in coupling
the BCR to the cytoskeleton, or in controlling vesicular transport.
Lastly, it has been proposed that they function as chaperones,
helping in the folding and assembly of transmembrane proteins.
Relevant Literature
[0060] The B-cell receptor, its components, and its association
with the five immunoglobulin classes have been described by
Wienands et al., EMBO J. 9(2): 449-455 (1990), Venkitaraman et al.,
Nature 352: 777-781 (1991), Herren et al., Immunologic Res.
26(1-3): 35-43 (2002). BCR associated proteins have been described
by Adachi et al., EMBO J 15(7): 1534-1541 (1996) and Schamel et
al., PNAS 100(17): 9861-9866 (2003). The influence of the B-cell
receptor on B-cell development and survival has been described by
Reth, Annual Reviews of Immunology 10: 97-121 (1992), Kraus et al.,
Cell 117(6): 787-800 (2004), Sayegh et al., Immunological Reviews
175: 187-200 (2000), Reichlin et al, Journal of Experimental
Medicine 193(1): 13-23 (2001), Pike et al., Journal of Immunology
172: 2210-2218 (2004), Pelanda et al., Journal of Immunology 169:
865-872 (2002). Regulation of BCR signaling and its influence in
B-cell development and apoptosis have been described in Cronin et
al., J. Immunology 161: 252-259 (1998), Muller et al., PNAS 97
(15): 8451-8454 (2000), Cragg et al., Blood 100: 3068-3076 (2002),
Wang et al., J. Immunology 171: 6381-6388 (2003), Fuentes-Panana et
al., J. Immunology 174: 1245-1252 (2005). The disclosures of the
above cited references are incorporated herein by reference in
their entirety.
[0061] The present invention therefore is directed to methods for
co-expressing human(ized) Ig.alpha. and/or human(ized) Ig.beta. in
B-cells, particularly in transgenic animals that are capable of
producing a diversified human(ized) antibody repertoire to improve
B-cell survival in such transgenic animals. Types of animals
include larger non-human animals like rabbits, birds, chickens,
sheep, goats, cows, swine, horses and donkeys. When these animals
express an Ig translocus, because of their larger size, their
antibody yields should also be greater. Thus, this invention aims
at creating larger founder animals producing higher amounts
human(ized) immunoglobulins through enhanced B-cell development and
survival.
[0062] Accordingly, the present invention is directed to transgene
constructs encoding full-length human Ig.alpha. and Ig.beta.
polypeptides, or, chimeric transgene constructs encoding for
chimeric or humanized Ig.alpha. and chimeric or humanized Ig.beta.
polypeptides, as defined further below.
[0063] By "transgene or transgene construct encoding the human
Ig.alpha. and/or Ig.beta. polypeptide" is meant the native, full
length, human Ig.alpha. and/or Ig.beta. DNA sequence respectively,
as well as any variant, codon optimized DNA sequence which encodes
for a functionally equivalent polypeptide of Ig.alpha. or Ig.beta.,
but which has a different DNA sequence based on codon degeneracy.
This concept is discussed in detail further below. The native, full
length, human Ig.alpha. polypeptide sequence is defined in SEQ ID
NO: 1 (FIG. 1). The native, full length, human Ig.beta. polypeptide
sequence is defined in SEQ ID NO: 7 (FIG. 1).
[0064] Also referred to herein is "nucleic acid molecule or
transgene or transgene construct encoding the chimeric or
human(ized) Ig.alpha.". A "chimeric Ig.alpha." subunit or protein
or polypeptide refers to an Ig.alpha. polypeptide sequence from an
animal (e.g.; rat, mouse, human, rabbit, chicken, etc.), in which
one or more domains of the Ig.alpha. polypeptide are replaced with
a corresponding domain or domains from a different Ig.alpha.
polypeptide of another animal or species, or with a corresponding
domain or domains from a different allelic Ig.alpha. version, or
from a variant Ig.alpha. sequence with one or more amino acid
substitutions, or from a variant Ig.alpha. sequence having at least
85% sequence identity to the corresponding domain of a given
Ig.alpha. sequence. The terms "chimeric Ig.alpha." and "human(ized)
Ig.alpha." are used interchangeably throughout the specification.
The non-human Ig.alpha. polypeptide sequences from which the
intracellular and/or the transmembrane domain sequences can be
obtained, for example, include, but are not limited to, bovine (SEQ
ID NO: 2); murine (SEQ ID NO: 3); canine (SEQ ID NO: 4); primate
((SEQ ID NO: 5); rabbit (SEQ ID NO: 6) or other non-human
sequences.
[0065] Also referred to herein is "nucleic acid molecule or
transgene or transgene construct encoding the chimeric or
human(ized) Ig.beta.". A "chimeric Ig.beta." subunit or protein or
polypeptide refers to an Ig.beta. polypeptide sequence from an
animal (e.g.; rat, mouse, human, rabbit, chicken, etc.), in which
one or more domains of the Ig.beta. polypeptide are replaced with a
corresponding domain or domains from a different Ig.beta.
polypeptide of another animal or species, or with a corresponding
domain or domains from a different allelic Ig.beta. version, or
from a variant Ig.beta. sequence with one or more amino acid
substitutions, or from a variant Ig.beta. sequence having at least
85% sequence identity to the corresponding domain of a given
Ig.beta. sequence. The terms "chimeric Ig.beta." and "human(ized)
Ig.beta." are used interchangeably throughout the specification.
The non-human Ig.beta. polypeptide sequences from which the
intracellular and/or the transmembrane domain sequences can be
obtained, for example, include, but are not limited to, canine (SEQ
ID NO: 8); rat (SEQ ID NO: 9); bovine (SEQ ID NO: 10); murine (SEQ
ID NO: 11); chicken (SEQ. ID NO: 12); or other non-human
sequences.
[0066] Thus, briefly, a chimeric Ig.alpha. or Ig.beta. transgene
consists of 1) a nucleotide sequence encoding the extracellular
domain of the human Ig.alpha. or Ig.beta. respectively, and 2) a
nucleotide sequence encoding the transmembrane and the
intracellular domain of the Ig.alpha. or Ig.beta. from the host
transgenic animal, respectively.
[0067] In a further aspect, the present invention is also directed
to transgenic constructs encoding for a human(ized) immunoglobulins
or locii as described in a previously filed U.S. applications, now
available as U.S. Publication No. 2003-0017534, published Jan. 23,
2003 and U.S. Publication No. 2006-0026696, published Feb. 2, 2006,
the disclosures of which is hereby incorporated by reference in
their entirety. The transgenic animals, B-cells or cell lines
generated thereof, and the relevant methodologies disclosed therein
also form an aspect of this invention.
[0068] In an alternative approach to the above mentioned aspect,
the present invention is also directed to transgenic constructs
encoding for human(ized) immunoglobulin or Ig chain or loci, as
described in U.S. Pat. No. 5,545,806, issued August 1996; U.S. Pat.
No. 5,545,807, issued August 1996 and U.S. Pat. No. 5,569,825,
issued October 1996, U.S. Pat. No. 7,064,244, issued Jun. 20, 2006;
or in PCT Publication Nos. WO 92/03918, WO 02/12437, and in U.S.
Pat. Nos. 5,814,318, and 5,570,429; also see Jakobovits et al.,
Proc. Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al.,
Nature, 362: 255 (1993); Bruggemann et al., Year in Immunol., 7: 33
(1993); Pluschke et al., Journal of Immunological Methods 215:
27-37 (1998); Neuberger et al., Nature 338: 350-2 (1989); Lonberg
et al., Int. Rev. Immunol. 13(1):65-93 (1995); and Bruggemann et
al., Curr. Opin. Biotechnol., 8(4): 455-8 (1997); and Kuroiwa et
al., Nature Biotech 20(9): 889-894 (2002) the disclosures of which
is hereby incorporated by reference in their entirety. The
transgenic animals, B-cells or cell lines generated thereof, and
the relevant methodologies disclosed therein also form an aspect of
this invention.
[0069] The transgenes or transgene constructs may be introduced
into the animal's genome by a variety of techniques including
microinjection of pronuclei, transfection, nuclear transfer
cloning, sperm-mediated gene transfer, testis-mediated gene
transfer, and the like.
[0070] In one embodiment, the human Ig.alpha. and/or Ig.beta. gene,
is preferably expressed in the B-cells of the transgenic animal by
means of an immune-specific promoter, preferably a B-cell specific
promoter. This human Ig.alpha. or Ig.beta. gene expression happens
preferably within B-cells alone, leading to enhanced B-cell
development and survival of the non-human transgenic animal. By
"B-cell specific promoter" is meant the promoter/enhancers sequence
of any B-cell specific genes, and/or variants or engineered
portions thereof, that normally controls the expression of genes
expressed in a B-cell, examples of which include, but are not
limited to, promoters/enhancers of CD19, CD20, CD21, CD22, CD23,
CD24, CD40, CD72, Blimp-1, CD79b (also known as B29 or Ig beta),
mb-1 (also known as Ig alpha), tyrosine kinase blk, VpreB,
immunoglobulin heavy chain, immunoglobulin kappa light chain,
immunoglobulin lambda-light chain, immunoglobulin J-chain, etc. In
a preferred embodiment, the CD79a, CD79b, or kappa light chain
promoter/enhancer drives the B-cell specific expression of the
human Ig.alpha. and/or Ig.beta. genes.
[0071] In yet another embodiment, the transgene construct
comprising the nucleic acid molecule encoding the human Ig.alpha.
and/or Ig.beta. genes is coexpressed with the transgene construct
comprising an exogenous immunoglobulin or immunoglobulin (Ig) chain
transgene locus. In this embodiment, both the Ig transgene locus
and the human Ig.alpha. and/or Ig.beta. transgene may be present on
the same transgenic expression vector or on two different
transgenic expression vectors. In the latter case, the two
transgenic expression vectors may be introduced into the non-human
transgenic animal either at the same time or sequentially.
[0072] In accordance with this invention, variants of the human
full length or extracellular domain alone, of Ig.alpha. or Ig.beta.
are included herein. By this is meant nucleic acid sequences that
allow for the degeneracy of the genetic code, nucleic acid
sequences that encode for a polypeptide sequence that comprises
amino acid substitutions of functionally equivalent residues and/or
mutations that enhance the functionality of the extracellular
domain. "Functionality of the extracellular domain" includes, but
is not limited to, formation of a BCR capable of signal
transduction
[0073] By allowing for the degeneracy of the genetic code, the
invention encompasses sequences that have at least about 70%, more
usually about 80 to 85%, preferably at least about 90% and most
preferably about 95% sequence identity to the extracellular
polypeptide sequence of human Ig.alpha. and human Ig.beta..
[0074] The term biologically functional equivalent is well
understood in the art and is further defined in detail herein.
Accordingly, sequences that have between about 70% and about 80%;
or more preferably, between about 81% and about 90%; or even more
preferably, between about 91% and about 99% identical at the amino
acid level are considered functionally equivalent to human
Ig.alpha. and Ig.beta., provided the biological activity of the
proteins is maintained.
[0075] The term functionally equivalent codon is used herein to
refer to codons that encode the same amino acid, such as the six
codons for arginine or serine, and also refers to codons that
encode biologically equivalent amino acids.
[0076] The following is a discussion based upon changing of the
amino acids of a protein to create an equivalent, or even an
improved, second-generation molecule. For example, certain amino
acids may be substituted for other amino acids in a protein
structure without appreciable loss of interactive binding capacity
with structures such as, for example, antigen-binding regions of
antibodies or binding sites on substrate molecules. Since it is the
interactive capacity and nature of a protein that defines that
protein's biological functional activity, certain amino acid
substitutions can be made in a protein sequence, and in its
underlying DNA coding sequence, and nevertheless produce a protein
with like properties. It is thus contemplated by the inventors that
various changes may be made in the DNA sequences of genes without
appreciable loss of their biological utility or activity, as
discussed below.
[0077] In making such changes, the hydropathic index of amino acids
may also be considered. The importance of the hydropathic amino
acid index in conferring interactive biologic function on a protein
is generally understood in the art (Kyte & Doolittle, 1982). It
is accepted that the relative hydropathic character of the amino
acid contributes to the secondary structure of the resultant
protein, which in turn defines the interaction of the protein with
other molecules, for example, enzymes, substrates, receptors, DNA,
antibodies, antigens, and the like.
[0078] It also is understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with a biological property of the protein. As
detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity
values have been assigned to amino acid residues: arginine (+3.0);
lysine (+3.0); aspartate (+3.0.+-0.1); glutamate (+3.0.+-0.1);
serine (+0.3); asparagine (+0.2) glutamine (+0.2); glycine (0);
threonine (-0.4); proline (-0.5.+-0.1); alanine (-0.5); histidine
(-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine
(-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5);
tryptophan (-3.4).
[0079] It is understood that an amino acid can be substituted for
another having a similar hydrophilicity value and still produce a
biologically equivalent and immunologically equivalent protein. In
such changes, the substitution of amino acids whose hydrophilicity
values are within .+-0.2 is preferred, those that are within .+-0.1
are particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0080] As outlined herein, amino acid substitutions generally are
based on the relative similarity of the amino acid side-chain
substituents, for example, their hydrophobicity, hydrophilicity,
charge, size, and the like. Exemplary substitutions that take into
consideration the various foregoing characteristics are well known
to those of skill in the art and include: arginine and lysine;
glutamate and aspartate; serine and threonine; glutamine and
asparagine; and valine, leucine and isoleucine.
[0081] Another embodiment for the preparation of polypeptides
according to the invention is the use of peptide mimetics. Mimetics
are peptide-containing molecules that mimic elements of protein
secondary structure (Johnson 1993). The underlying rationale behind
the use of peptide mimetics is that the peptide backbone of
proteins exists chiefly to orient amino acid side chains in such a
way as to facilitate molecular interactions, such as those of
antibody and antigen. A peptide mimetic is expected to permit
molecular interactions similar to the natural molecule. These
principles may be used, in conjunction with the principles outlined
above, to engineer second generation molecules having many of the
natural properties of human Ig.alpha. or Ig.beta. with altered and
improved characteristics.
[0082] Thus, variant nucleic acid sequences that encode for human
Ig.alpha. or Ig.beta. and functionally equivalent polypeptides of
human Ig.alpha. or Ig.beta. are useful in this invention.
[0083] The transgenic vectors containing the genes of interest may
be introduced into the recipient cell or cells and then integrated
into the genome of the recipient cell or cells by random
integration or by targeted integration.
[0084] For random integration, a transgenic vector containing a
human Ig.alpha. or Ig.beta. can be introduced into an animal
recipient cell by standard transgenic technology. For example, a
transgenic vector can be directly injected into the pronucleus of a
fertilized oocyte. A transgenic vector can also be introduced by
co-incubation of sperm with the transgenic vector before
fertilization of the oocyte. Transgenic animals can be developed
from fertilized oocytes. Another way to introduce a transgenic
vector is by transfecting embryonic stem cells and subsequently
injecting the genetically modified embryonic stem cells into
developing embryos. Alternatively, a transgenic vector (naked or in
combination with facilitating reagents) can be directly injected
into a developing embryo. Ultimately, chimeric transgenic animals
are produced from the embryos which contain the human(ized) Ig
transgene integrated in the genome of at least some somatic cells
of the transgenic animal.
[0085] In a particular embodiment, a transgene containing a human
Ig.alpha. or Ig.beta. is randomly integrated into the genome of
recipient cells (such as fertilized oocyte or developing embryos)
derived from animal strains with an impaired expression of
endogenous Ig.alpha. or Ig.beta.. The use of such animal strains
permits preferential expression of immunoglobulin molecules from
the human(ized) transgenic Ig locus. Alternatively, transgenic
animals with human(ized) Ig.alpha. and/or Ig.beta. transgenes can
be mated with animal strains with impaired expression of endogenous
Ig.alpha. and/or Ig.beta.. Offspring homozygous for impaired
Ig.alpha. and/or Ig.beta. and human(ized) Ig.alpha. and/or Ig.beta.
can be obtained. Alternatively, expression of endogenous Ig.alpha.
and/or Ig.beta. may be inhibited or lowered using antisense
technology, intracellular anti-Ig.alpha. and/or Ig.beta.
expression, and the like. In one embodiment, the method of choice
for the knocking down the endogenous production of Ig.alpha. and/or
Ig.beta. of the host animal is the RNA interference (RNA.sub.i)
method, which introduces either double-stranded RNA (ds RNA) or
more preferably, short or small interfering RNA duplexes (siRNA)
into the B-cells having intracellular host animal Ig.alpha. and/or
Ig.beta. nucleic acid sequences. This can be achieved using
commercially available kits, including but not limited to, Block
iT.TM. or Stealth.TM. RNA kits from Invitrogen Corp.
[0086] For targeted integration, a transgenic vector can be
introduced into appropriate animal recipient cells such as
embryonic stem cells or already differentiated somatic cells.
Afterwards, cells in which the transgene has integrated into the
animal genome and has replaced the corresponding endogenous gene by
homologous recombination can be selected by standard methods (See
for example, Kuroiwa et al, Nature Genetics 2004, June 6). The
selected cells may then be fused with enucleated nuclear transfer
unit cells, e.g. oocytes or embryonic stem cells, which are
totipotent and capable of forming a functional neonate. Fusion is
performed in accordance with conventional techniques which are well
established. Enucleation of oocytes and nuclear transfer can also
be performed by microsurgery using injection pipettes. (See, for
example, Wakayama et al., Nature (1998) 394:369.) The resulting egg
cells are then cultivated in an appropriate medium, and transferred
into synchronized recipients for generating transgenic animals.
Alternatively, the selected genetically modified cells can be
injected into developing embryos which are subsequently developed
into chimeric animals.
[0087] Further, according to the present invention, a transgenic
animal capable of producing human(ized) Ig.alpha. and/or Ig.beta.
can also be made by introducing into a recipient cell or cells, one
or more of the recombination vectors described herein above, one of
which carries a human Ig.alpha. and/or Ig.beta. gene segment,
linked to 5' and 3' flanking sequences that are homologous to the
flanking sequences of the endogenous Ig.alpha. and/or Ig.beta. gene
segment, then selecting cells in which the endogenous Ig.alpha.
and/or Ig.beta. gene segment is replaced by the human Ig.alpha.
and/or Ig.beta. gene segment by homologous recombination, and
deriving an animal from the selected genetically modified recipient
cell or cells.
[0088] Similar to the target insertion of a transgenic vector,
cells appropriate for use as recipient cells in this approach
include embryonic stem cells or already differentiated somatic
cells. A recombination vector carrying a human Ig.alpha. and/or
Ig.beta. gene segment can be introduced into such recipient cells
by any feasible means, e.g., transfection. Afterwards, cells in
which the human Ig.alpha. and/or Ig.beta. gene segment has replaced
the corresponding endogenous Ig.alpha. and/or Ig.beta. gene segment
by homologous recombination, can be selected by standard methods.
These genetically modified cells can serve as nuclei donor cells in
a nuclear transfer procedure for cloning a transgenic animal.
Alternatively, the selected genetically modified embryonic stem
cells can be injected into developing embryos which can be
subsequently developed into chimeric animals.
[0089] In a specific embodiment, the transgene constructs of the
invention may be introduced into the transgenic animals during
embryonic life by directly injecting the transgenes into the embryo
or indirectly by injecting them into the pregnant mother or into
the egg-laying hen. Transgenic animals produced by any of the
foregoing methods form another embodiment of the present invention.
The transgenic animals have at least one, i.e., one or more,
human(ized) Ig.alpha. and/or Ig.beta. gene in the genome, from
which a functional human(ized) Ig.alpha. and/or Ig.beta. protein
can be produced.
[0090] Further, the transgene constructs of the invention, namely,
the human or humanized Ig.alpha. and/or Ig.beta. transgene and the
humanized immunoglobulin transgene, are preferably expressed
against a knockout background of either one, or more preferably
both, the endogenous Ig, as well as the endogenous Ig.alpha. and/or
Ig.beta. knockouts. Thus the transgenic animals of the present
invention are capable of rearranging the human(ized) Ig loci and
efficiently expressing a functional repertoire of human(ized)
antibodies against a background that has substantially reduced
endogenous Ig expression and more preferably, substantially reduced
endogeous Ig.alpha. and/or Ig.beta. as well. In this context, by
"substantially" is meant the degree of endogenous production, of
either endogenous Ig expression alone or additionally, endogeous
Ig.alpha. and/or Ig.beta. expression is reduced preferably at least
about 30%-49%, or more preferably at least about 50%-79%, or even
more preferably at least about 80-89%, or most preferably by about
90-100%.
[0091] The present invention provides transgenic rabbits expressing
one or more human(ized) Ig loci and human(ized) Ig.alpha. and/or
Ig.beta., that are capable of rearranging and gene converting the
human(ized) Ig loci, and expressing a functional repertoire of
human(ized) antibodies. Preferably, these rabbits, additionally, do
not produce substantial amounts of functional. endogenous, rabbit
immunoglobulins or functional endogenous, rabbit Ig.alpha. and/or
Ig.beta..
[0092] The present invention also provides other large transgenic
animals, including but not limited to, birds, rodents and farm
animals like cows, pigs, sheep, goats, donkeys, horses and the like
expressing one or more human(ized) Ig loci and human(ized)
Ig.alpha. and/or Ig.beta.. Again, preferably, these animals,
additionally, do not produce substantial amounts of functional.
endogenous, immunoglobulins or functional endogenous, Ig.alpha.
and/or Ig.beta.. Thus, these transgenic animals are capable of
rearranging the human(ized) Ig loci and efficiently expressing a
functional repertoire of human(ized) antibodies, with increased
yields.
[0093] The invention is also directed to B-cells isolated from the
different types of transgenic animals described above, that express
the human(ized) Ig.alpha. and/or Ig.beta. gene and the human(ized)
immunoglobulin loci. Further, such B-cells can be immortalized
using conventional methods known and used by skilled artisans in
the field, including but not limited to, using viral
transformation, etc.
[0094] Immunization with antigen leads to the production of
human(ized) antibodies against the same antigen in said transgenic
animals.
[0095] Although preferred embodiments of the present invention are
directed to transgenic animals having human(ized) Ig loci, it is to
be understood that transgenic animals having primatized Ig loci and
primatized polyclonal antisera are also within the spirit of the
present invention. Similar to human(ized) polyclonal antisera
compositions, primatized polyclonal antisera compositions are
likely to have a reduced immunogenicity in human individuals.
[0096] Once a transgenic non-human animal capable of producing
diversified human(ized) immunoglobulin molecules is made (as
further set forth below), human(ized) immunoglobulins and
human(ized) antibody preparations against an antigen can be readily
obtained by immunizing the animal with the antigen. A variety of
antigens can be used to immunize a transgenic host animal. Such
antigens include, microorganism, e.g. viruses and unicellular
organisms (such as bacteria and fungi), alive, attenuated or dead,
fragments of the microorganisms, or antigenic molecules isolated
from the microorganisms.
[0097] Preferred bacterial antigens for use in immunizing an animal
include purified antigens from Staphylococcus aureus such as
capsular polysaccharides type 5 and 8, recombinant versions of
virulence factors such as alpha-toxin, adhesin binding proteins,
collagen binding proteins, and fibronectin binding proteins.
Preferred bacterial antigens also include an attenuated version of
S. aureus, Pseudomonas aeruginosa, enterococcus, enterobacter, and
Klebsiella pneumoniae, or culture supernatant from these bacteria
cells. Other bacterial antigens which can be used in immunization
include purified lipopolysaccharide (LPS), capsular antigens,
capsular polysaccharides and/or recombinant versions of the outer
membrane proteins, fibronectin binding proteins, endotoxin, and
exotoxin from Pseudomonas aeruginosa, enterococcus, enterobacter,
and Klebsiella pneumoniae.
[0098] Preferred antigens for the generation of antibodies against
fungi include attenuated version of fungi or outer membrane
proteins thereof, which fungi include, but are not limited to,
Candida albicans, Candida parapsilosis, Candida tropicalis, and
Cryptococcus neoformans.
[0099] Preferred antigens for use in immunization in order to
generate antibodies against viruses include the envelop proteins
and attenuated versions of viruses which include, but are not
limited to respiratory synctial virus (RSV) (particularly the
F-Protein), Hepatitis C virus (HCV), Hepatits B virus (HBV),
cytomegalovirus (CMV), EBV, and HSV.
[0100] Therapeutic antibodies can be generated for the treatment of
cancer by immunizing transgenic animals with isolated tumor cells
or tumor cell lines; tumor-associated antigens which include, but
are not limited to, Her-2-neu antigen (antibodies against which are
useful for the treatment of breast cancer); CD19, CD20, CD22 and
CD53 antigens (antibodies against which are useful for the
treatment of B-cell lymphomas), (3) prostate specific membrane
antigen (PMSA) (antibodies against which are useful for the
treatment of prostate cancer), and 17-1A molecule (antibodies
against which are useful for the treatment of colon cancer).
[0101] The antigens can be administered to a transgenic host animal
in any convenient manner, with or without an adjuvant, and can be
administered in accordance with a predetermined schedule.
[0102] After immunization, serum or milk from the immunized
transgenic animals can be fractionated for the purification of
pharmaceutical grade polyclonal antibodies specific for the
antigen. In the case of transgenic birds, antibodies can also be
made by fractionating egg yolks. A concentrated, purified
immunoglobulin fraction may be obtained by chromatography
(affinity, ionic exchange, gel filtration, etc.), selective
precipitation with salts such as ammonium sulfate, organic solvents
such as ethanol, or polymers such as polyethyleneglycol.
[0103] The fractionated human(ized) antibodies may be dissolved or
diluted in non-toxic, non-pyrogenic media suitable for intravenous
administration in humans, for instance, sterile buffered
saline.
[0104] The antibody preparations used for administration are
generally characterized by having immunoglobulin concentrations
from 0.1 to 100 mg/ml, more usually from 1 to 10 mg/ml. The
antibody preparation may contain immunoglobulins of various
isotypes. Alternatively, the antibody preparation may contain
antibodies of only one isotype, or a number of selected
isotypes.
[0105] For making a human(ized) monoclonal antibody, spleen cells
are isolated from the immunized transgenic animal whose B-cells
expressing the animal's endogenous immunoglobulin have been
depleted. Isolated spleen cells are used either in cell fusion with
transformed cell lines for the production of hybridomas, or cDNAs
encoding antibodies are cloned by standard molecular biology
techniques and expressed in transfected cells. The procedures for
making monoclonal antibodies are well established in the art. See,
e.g., European Patent Application 0 583 980 A1 ("Method For
Generating Monoclonal Antibodies From Rabbits"), U.S. Pat. No.
4,977,081 ("Stable Rabbit-Mouse Hybridomas And Secretion Products
Thereof"), WO 97/16537 ("Stable Chicken B-cell Line And Method of
Use Thereof"), and EP 0 491 057 B1 ("Hybridoma Which Produces Avian
Specific Immunoglobulin G"), the disclosures of which are
incorporated herein by reference. In vitro production of monoclonal
antibodies from cloned cDNA molecules has been described by
Andris-Widhopf et al., "Methods for the generation of chicken
monoclonal antibody fragments by phage display", J Immunol Methods
242:159 (2000), and by Burton, D. R., "Phage display",
Immunotechnology 1:87 (1995), the disclosures of which are
incorporated herein by reference.
[0106] In most instances the antibody preparation consists of
unmodified immunoglobulins, i.e., human(ized) antibodies prepared
from the animal without additional modification, e.g., by chemicals
or enzymes. Alternatively, the immunoglobulin fraction may be
subject to treatment such as enzymatic digestion (e.g. with pepsin,
papain, plasmin, glycosidases, nucleases, etc.), heating, etc,
and/or further fractionated to generate "antibody fragments".
[0107] The present invention also includes pharmaceutical
compositions or antibody preparations comprising the antibodies or
their fragments obtained by the methods defined above The term
"pharmaceutically acceptable ingredient" or "formulation" as used
herein is intended to encompass a product comprising the claimed
active ingredient(s), namely human(ized) antibody or antibody
fragment, in specified amounts, as well as any product which
results, directly or indirectly, from the combination of the
specified active ingredient(s) in the specified amounts. Such term
is intended to encompass a product comprising the active
ingredient(s), and the inert ingredient(s) that make up the
carrier, as well as any product which results, directly or
indirectly, from combination, complexation or aggregation of any
two or more of the ingredients, or from dissociation of one or more
of the ingredients, or from other types of reactions or
interactions of one or more of the ingredients. Accordingly, the
"pharmaceutical compositions" of the present invention encompass
any composition made by admixing any active compound of the present
invention and a pharmaceutically acceptable carrier.
[0108] The terms "administration of" and or "administering a"
compound should be understood to mean providing any active compound
of the invention, in any formulation, to an individual in need of
treatment.
[0109] The pharmaceutical compositions for the administration of
the compounds of this invention may conveniently be presented in
dosage unit form and may be prepared by methods well known in the
art of pharmacy. Suitable methods and carriers for use are those
that are well-described in the art, and for example, in Remington,
The Science and Practice of Pharmacy, ed. Gennaro et al., 20th Ed.
(2000), although the skilled artisan in the field of immunology
will readily recognize that other methods are known and are
suitable for preparing the compositions of the present invention.
All methods include the step of bringing the active ingredient into
association with the carrier which constitutes one or more
accessory ingredients. In general, the pharmaceutical compositions
are prepared by uniformly and intimately bringing the active
ingredient into association with a liquid carrier or a finely
divided solid carrier or both, and then, if necessary, shaping the
product into the desired formulation. In the pharmaceutical
composition the active ingredient is included in an effective
amount, discussed above, sufficient to produce the desired effect
upon the process or condition of diseases. Furthermore,
formulations for the controlled, prolonged release of antibody
molecules have been described in U.S. Pat. No. 6,706,289, whose
methods are incorporated by reference herein.
[0110] Thus, the transgenic constructs, the vectors comprising the
transgene constructs and the transgenic animals generated using the
methods described above are all embodiments of the invention.
[0111] The invention is further illustrated, but by no means
limited, by the following examples.
EXAMPLE 1
Transfection of a Rabbit B-Cell Line with Human Ig.alpha. and
Ig.beta.
[0112] To demonstrate the effect of human Ig.alpha. and Ig.beta. on
the expression of human mIgM in rabbit B-cells, such cells are
transfected with expression vectors encoding human Ig.alpha. or
Ig.beta. or a human mIgM.
[0113] Human Ig.alpha. and human Ig.beta. and human IgM encoding
genes are cloned in expression vectors.
[0114] An immortalized rabbit B-cell line is transfected with the
expression vectors and cultured in medium in the presence of
neomycin for the selection of stable transfectants. Resistant cells
are analyzed by flow cytometry using antibodies specific for human
IgM and human Ig.alpha. and/or Ig.beta.. Transfection of rabbit
B-cells with an expression vector encoding human IgM results in low
cell surface expression of human IgM. Cotransfection with human
Ig.alpha. and/or Ig.beta. results in high cell surface expression
of human mIgM. This demonstrates that human Ig.alpha. and/or .beta.
is necessary and sufficient for high cell surface expression of
human(ized) or chimeric mIgM.
EXAMPLE 2
Transfection of a B-Cell Line Derived From Any Animal With Human
Ig.alpha. and Ig.beta.
[0115] To demonstrate the effect of human Ig.alpha. and Ig.beta. on
the expression of human mIgM in animal derived B-cells, expression
vectors encoding human Ig.alpha. or Ig.beta. or a human mIgM are
transfected in B-cell derived from chicken (DT40), cow, and
pigs.
[0116] Immortalized B-cell lines are transfected with the
expression vectors and cultured in medium in the presence of
neomycin for the selection of stable transfectants. Resistant cells
are analyzed by flow cytometry using antibodies specific for human
IgM and human Ig.alpha. and/or Ig.beta.. Transfection of rabbit
B-cells with an expression vector encoding human IgM results in low
cell surface expression of human IgM. Cotransfection with human
Ig.alpha. and/or Ig.beta. results in high cell surface expression
of human mIgM. This demonstrates that human Ig.alpha. and/or
Ig.beta., is necessary and sufficient for high cell surface
expression of human(ized) or chimeric mIgM.
EXAMPLE 3
Transgenic Rabbits Expressing the Humanized Immunoglobulin Light
and/or Heavy Chain Transgene With or Without Human Ig.alpha. and/or
Ig.beta.
[0117] Transgenic rabbits were generated as described by Fan et al.
(Pathol. Int. 49: 583-594, 1999). Briefly, female rabbits were
superovulated using standard methods and mated with male rabbits.
Pronuclear-stage zygotes were collected from oviduct and placed in
an appropriate medium such as Dulbecco's phosphate buffered saline
supplemented with 20% fetal bovine serum. The exogenous DNA (e.g.,
expression vectors containing human(ized) immunoglobulin locus or
human Ig.alpha. or human Ig.beta.) were microinjected into the
pronucleus with the aid of a pair of manipulators. Morphological
surviving zygotes were transferred to the oviducts of
pseudopregnant rabbits. Pseudopregnancy was induced by the
injection of human chorionic gonadotrophin (hCG). Between about
0.1-1% of the injected zygotes developed into live transgenic
rabbits. Integration of the transgene in the genome was confirmed
by PCR and FISH.
[0118] The presence of antibodies containing human IgG and/or human
kappa light chain antigenic determinants in the serum of transgenic
founder rabbits was determined using an ELISA assay. Antibody
expression on the surface of B-cells was analyzed by flow
cytometry. Rabbits with a transgene encoding a human(ized)
immunoglobulin heavy chain locus, expressed 1-10 ug/ml human IgM.
Young animals (6-9 weeks) expressed 100-4000 ug/ml human IgG.
However, the expression of human IgG declined rapidly to levels of
10-100 ug/ml. Flow cytometric analysis of B-cells in peripheral
blood revealed a small population of human mIgM+ cells (1-2%). The
appendix of young rabbits contained up to 10% human mIgM+ cells
which disappeared rapidly with age.
[0119] Introduction of transgenes encoding human Ig.alpha. and/or
Ig.beta. results in the expression of 100-2000 ug/ml human(ized)
IgM in serum and stable expression of 2000-12000 ug/ml human(ized)
IgG. In appendix 30-70% of lymphocytes are human(ized) mIgM+. In
peripheral blood equivalent numbers of B-cells express rabbit and
human(ized) mIgM or mIgG.
[0120] All references cited throughout the disclosure along with
references cited therein are hereby expressly incorporated by
reference.
[0121] While the invention is illustrated by reference to certain
embodiments, it is not so limited. One skilled in the art will
understand that various modifications are readily available and can
be performed without substantial change in the way the invention
works. All such modifications are specifically intended to be
within the scope of the invention claimed herein.
Sequence CWU 1
1
121226PRTHomo sapien 1Met Pro Gly Gly Pro Gly Val Leu Gln Ala Leu
Pro Ala Thr Ile Phe1 5 10 15 Leu Leu Phe Leu Leu Ser Ala Val Tyr
Leu Gly Pro Gly Cys Gln Ala 20 25 30 Leu Trp Met His Lys Val Pro
Ala Ser Leu Met Val Ser Leu Gly Glu 35 40 45 Asp Ala His Phe Gln
Cys Pro His Asn Ser Ser Asn Asn Ala Asn Val 50 55 60 Thr Trp Trp
Arg Ile Leu His Gly Asn Tyr Thr Trp Pro Pro Glu Phe65 70 75 80 Leu
Gly Pro Gly Glu Asp Pro Asn Gly Thr Leu Ile Ile Gln Asn Val 85 90
95 Asn Lys Ser His Gly Gly Ile Tyr Val Cys Arg Val Gln Glu Gly Asn
100 105 110 Glu Ser Tyr Gln Gln Ser Cys Gly Thr Tyr Leu Arg Val Arg
Gln Pro 115 120 125 Pro Pro Arg Pro Phe Leu Asp Met Gly Glu Gly Thr
Lys Asn Arg Ile 130 135 140 Ile Thr Ala Glu Gly Ile Ile Leu Leu Phe
Cys Ala Val Val Pro Gly145 150 155 160 Thr Leu Leu Leu Phe Arg Lys
Arg Trp Gln Asn Glu Lys Leu Gly Leu 165 170 175 Asp Ala Gly Asp Glu
Tyr Glu Asp Glu Asn Leu Tyr Glu Gly Leu Asn 180 185 190 Leu Asp Asp
Cys Ser Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly 195 200 205 Thr
Tyr Gln Asp Val Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu 210 215
220 Lys Pro225 265PRTBos taurus 2Glu Gln Ala Ser Pro Ile Ala Gly
Val Glu Trp Gly Pro Val Thr Val1 5 10 15 Glu Val Arg Leu Thr Gly
Thr His Val Gln Ser Ser Ser Val Met Tyr 20 25 30 Arg Gly Asp Val
Gly Ala Gly Glu Lys Pro Thr Arg Met Arg Gln Ser 35 40 45 Asp Lys
Lys Ile Arg Asp Leu Asn Ile Met Phe Ala Ile Gln Asp His 50 55 60
Ala65 366PRTMus musculus 3Leu Glu Ala Arg Leu Leu Leu Tyr Ala Cys
Arg Val Glu Gly Gly Pro1 5 10 15 Thr Asn Glu Arg Leu Thr Glu Asn
Gly Arg Pro Ile Phe Ser Gln Ser 20 25 30 Ile Val Pro Gln Gly Thr
Thr Gln Phe Phe Pro Glu Asn Arg Leu Trp 35 40 45 Gln Ile Asn Asn
Ile Leu Lys Arg Asn Val Phe Val Met Pro Asp Asn 50 55 60 His Ala65
466PRTCanis 4Leu Cys Thr Ile Gly Gly Ser Val Asp Gly Gly Pro Met
Thr Thr Arg1 5 10 15 Leu Leu Arg Arg Leu Ser Ser Lys Leu Ile Val
Gln Ala Asp Ile Ser 20 25 30 Tyr Lys Gly Glu Thr Asp Thr Met Met
Arg Gln Glu Lys Asp Leu Asn 35 40 45 Gln Lys Ile Leu Ser Ser Glu
Arg Leu Asn Met Phe Val Gln Asp His 50 55 60 Gly Asp65 573PRTPan
5Ser Ala Val Arg Glu Trp Pro Ser Pro Gly Pro Tyr Ser His Cys Pro1 5
10 15 Ala Gly Asp Thr Arg Phe Ile Phe Glu Val Gly Ile Glu Pro Ile
Pro 20 25 30 Ser Met Trp Val Ser Asn Arg Leu Gly Gln Arg Asp Gly
His Ser Pro 35 40 45 Leu Gln Lys Val Ser Pro Leu Gly Pro Leu Ser
Gln Pro Gly Glu Gly 50 55 60 Leu Gly Arg Gly Thr Pro Asn Ala Gln65
70 62PRTOryctolagus cuniculus 6Asp His1 7229PRTHomo sapien 7Met Ala
Arg Leu Ala Leu Ser Pro Val Pro Ser His Trp Met Val Ala1 5 10 15
Leu Leu Leu Leu Leu Ser Ala Glu Pro Val Pro Ala Ala Arg Ser Glu 20
25 30 Asp Arg Tyr Arg Asn Pro Lys Gly Ser Ala Cys Ser Arg Ile Trp
Gln 35 40 45 Ser Pro Arg Phe Ile Ala Arg Lys Arg Arg Phe Thr Val
Lys Met His 50 55 60 Cys Tyr Met Asn Ser Ala Ser Gly Asn Val Ser
Trp Leu Trp Lys Gln65 70 75 80 Glu Met Asp Glu Asn Pro Gln Gln Leu
Lys Leu Glu Lys Gly Arg Met 85 90 95 Glu Glu Ser Gln Asn Glu Ser
Leu Ala Thr Leu Thr Ile Gln Gly Ile 100 105 110 Arg Phe Glu Asp Asn
Gly Ile Tyr Phe Cys Gln Gln Lys Cys Asn Asn 115 120 125 Thr Ser Glu
Val Tyr Gln Gly Cys Gly Thr Glu Leu Arg Val Met Gly 130 135 140 Phe
Ser Thr Leu Ala Gln Leu Lys Gln Arg Asn Thr Leu Lys Asp Gly145 150
155 160 Ile Ile Met Ile Gln Thr Leu Leu Ile Ile Leu Phe Ile Ile Val
Pro 165 170 175 Ile Phe Leu Leu Leu Asp Lys Asp Asp Ser Lys Ala Gly
Met Glu Glu 180 185 190 Asp His Thr Tyr Glu Gly Leu Asp Ile Asp Gln
Thr Ala Thr Tyr Glu 195 200 205 Asp Ile Val Thr Leu Arg Thr Gly Glu
Val Lys Trp Ser Val Gly Glu 210 215 220 His Pro Gly Gln Glu225
851PRTCanis 8Gly Val His Asn Leu Val Gly Lys Thr His Gln Asp Thr
Gly Gly Ala1 5 10 15 Val Glu Ile Arg His Thr Lys Asp Val Ala Arg
Leu Glu Lys Pro Pro 20 25 30 Arg Asp Leu Leu Gln Asp Val Gln Ser
Ser Lys Gly Ser Phe Ser Lys 35 40 45 Arg Asp Asn 50 970PRTRattus
9Thr Val Cys Leu Met Phe Gly Met Thr Lys Ser Gln Pro Pro Ile Phe1 5
10 15 Gln Pro Lys His Ala Lys Ser Ser Met Phe His Thr Asp Tyr Val
Met 20 25 30 Thr Phe Arg Gln Lys Gly Asn Gln Arg Glu Phe Pro Asp
His Ile Ser 35 40 45 Gln Thr Arg Gly Val Tyr Leu Asn Gln Tyr Ser
Thr Glu Pro Asp Thr 50 55 60 Asp Leu Leu Asp Arg Asn65 70
1071PRTBos taurus 10Gly Ser Ile Gly Leu Asn Asn Leu Leu Gly Gly Lys
Leu Asp Lys Thr1 5 10 15 Asp Leu Leu Asp Asn Thr His Val Lys Gly
Ser Glu Ile Arg His Val 20 25 30 Glu Asp Asp Leu Phe Arg Pro Lys
Pro Ser Glu Lys Thr His Ala Gln 35 40 45 Ile Leu Gln His Lys Glu
Val Leu Ser Val Gln Gln Lys Glu Ala Lys 50 55 60 Gly Gln Arg Thr
Glu His Arg65 70 1170PRTMus musculus 11Thr Val Ser Met Cys Leu Leu
Phe Phe Gly Met Thr Ser Leu Pro Leu1 5 10 15 Phe Gln Pro Gln His
Ala Lys Ser Ser Met Phe Thr His Ala Leu Thr 20 25 30 Phe Arg Arg
Gly Ser Gln Gln Glu Val Ser Glu Ile Val Gln Thr Gly 35 40 45 Val
Tyr Asn Gln Tyr Lys Asp Ser Ala Asn His Asn Thr Asp Ser Leu 50 55
60 Leu Asp Arg Leu Gly Asn65 70 12139PRTGallus 12Met Gly Asp Phe
Cys Arg Arg Leu Trp Val Leu Gln Val Asn Trp Met1 5 10 15 Ala Ala
Ala Gly Gly Ile Pro Thr Asp Gly Asn Ser Thr Ser Arg Thr 20 25 30
Glu Val Gly Met His Tyr Ala Lys Asn Thr Ser His Phe Ile Thr Ser 35
40 45 Gln Pro His Ala Met Gln Tyr Lys Ala Leu Gly Asn Gly Lys Glu
Phe 50 55 60 His Val Asp Gln Ser Ser Asp Phe Ser Ile Asn Asn Thr
Asn Asp Arg65 70 75 80 Ile Ser Phe Ser Arg Ser Tyr Val Asp Ser Asn
Leu Thr Glu Glu Lys 85 90 95 Arg Gln Pro Asn Ser Ile Ser Arg Asn
Ile Gln Ile Gln Asn Thr Ile 100 105 110 Ile Leu Val Ile Ser Met Leu
Phe Glu Gly Arg Glu Arg Pro Glu Val 115 120 125 Glu Ile Thr Pro Phe
Asp Met Lys Ala Thr Glu 130 135
* * * * *